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Comparison of mixed enzymatic pretreatment and post-treatment for enhancing the cellulose nanofibrillation efficiency
Bioresource Technology 293 (2019) 122171
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Comparison of mixed enzymatic pretreatment and post-treatment for enhancing the cellulose nanofibrillation efficiency
T
Huiyang Biana,b, Maolin Dongb, Lidong Chenb, Xuelian Zhoua,b, Shuzhen Nia,b,c, Guigan Fangd, ⁎ Hongqi Daia,b, a
Jiangsu Co-Innovation Center of Efficient Processing and Utilization of Forest Resources, Nanjing Forestry University, Nanjing 210037, China College of Light Industry and Food Engineering, Nanjing Forestry University, Nanjing 210037, China c State Key Laboratory of Biobased Material and Green Papermaking, Qilu University of Technology, Shandong Academy of Sciences, Jinan 250353, China d China Institute of Chemical Industry of Forestry Products, Chinese Academy of Forestry, Nanjing 210042, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Endoglucanase Xylanase Enzymatic pretreatment Enzymatic post-treatment Cellulose nanofibrillation
In this work, lignocellulosic nanofibrils (LCNF) produced from mechanical fibrillation with mixed enzymatic pretreatment or post-treatment were compared and the chemical composition, water retention value (WRV), average-number height and crystallinity for the obtained LCNF were evaluated. Compared to pure mechanical fibrillation, both mixed enzymatic pretreatment and post-treatment could efficiently facilitate cellulose nanofibrillation. Moreover, mixed enzymatic pretreatment was more suitable for LCNF production, resulting in a relatively higher WRV of 909% and smaller average-number height of 15 nm. These discoveries provide new insights into a more efficient biological method for the production and application of cellulose nanomaterials.
1. Introduction Lignocellulosic biomass, as the most abundant carbon-neutral resource on the earth, is considered as a sustainable raw material used for the production of different high-value added products (e.g., cellulose nanomaterials, biofuels and chemicals) (Zhu et al., 2011). One promising new product is cellulose nanofibrils (CNF), which can be used in applications such as hydrogels, supercapacitors, biosensors and energy storage devices due to its high elastic modulus, high specific surface area, and low thermal expansion coefficient (Siró and Plackett, 2010). The most commonly used method to produce CNF is via pure mechanical fibrillation equipment including grinders, homogenizers and microfluidizers (Lavoine et al., 2012). However, the high energy consumption during mechanical treatment and the heterogeneity of products still limited its implementation (Nair et al., 2014). In order to address these issues, chemical or biological treatments has been employed to “open up/loose” the intrinsic recalcitrance of cellulosic fibers, thus decreasing the energy required for the fibrillation (Wang et al., 2018). Although various chemical pretreatment approaches (e.g., TEMPO-oxidation, carboxylation and sulfonation) could achieve the goal, the severe corrosion of equipment and the difficulties to recycle chemicals have limited their further application (Beltramino et al., 2016; Jiang and Hsieh, 2016; Wang et al., 2017). Comparing to the
⁎
chemical pretreatment approaches, biological treatment is considered to be more attractive due to the low enzyme loading, high selectivity of enzymatic treatment and the environmentally friendly reaction conditions. To date, several types of enzymatic pretreatments have been investigated to improve the cellulose nanofibrillation. Most of the works were focused on using canonical endoglucanase (EG), which could randomly cleavage the cellulose β-1,4 linkages at less organized region (Pääkkö et al., 2007). Other so-called auxiliary enzymes, such as hemicellulases, laccases and lytic polysaccharide monooxygenases (LPMO), even though did not directly hydrolyze cellulose, could selectively remove hemicellulose and lignin from the carbohydrate networks (Hu et al., 2018; Long et al., 2018). In addition, the synergistic cooperation between cellulases and xylanases could greatly open up the cellulosic fiber without sacrificing cellulose crystal structure and significantly enhance the cellulose nanofibrillation (Long et al., 2017; Tian et al., 2017). Recently, we and others have found endoglucanase posttreatment also could produce less entangled fibrils with thinner diameters (Bian et al., 2018; Wang et al., 2016). However, there are no publicly available researches focused on comparing the cellulose nanofibrillation efficiency using mixed enzymatic pretreatment and posttreatment. In this work, unbleached mixed hardwood pulp was pretreated or
Corresponding author. E-mail address: [email protected] (H. Dai).
https://doi.org/10.1016/j.biortech.2019.122171 Received 10 September 2019; Received in revised form 17 September 2019; Accepted 18 September 2019 Available online 20 September 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
Bioresource Technology 293 (2019) 122171
H. Bian, et al.
and after drying) to the dried substrate (Bian et al., 2017).
post-treated by two types of enzymes (endoglucanase and xylanase) to produce lignocellulosic nanofibrils (LCNF). Several physicochemical characteristics such as chemical composition, water retention value, fibril morphology and crystallinity were investigated to compare the mixed enzyme pretreatment and post-treatment on the cellulose nanofibrillation efficiency and LCNF properties.
2.5.3. Morphology analysis The morphologies of LCNF samples produced from mixed enzyme pretreatment and post-treatment were analyzed by atomic force microscopy (AFM, Dimension Edge, Bruker, Germany). Samples were diluted to solids consistency of 0.01 wt% and deposited onto clean mica substrates and air dried overnight at room temperature. AFM topographical images were obtained in tapping mode at 300 kHz using a standard silicon cantilever and a tip with radius of curvature of 8 nm. Fibril height distribution was measured using Gwyddion software (Department of Nanometrology, Czech Metrology Institute, Crezh Republic, 64-bit) and number-average fibril heights were calculated.
2. Materials and methods 2.1. Materials Two unbleached mixed hardwood pulps with lignin content of 17.2% and 3.9%, defined as H and L, were complimentarily provided from International Paper Company (Loveland, OH). These two pulp samples were acquired from the same mill but different production lines. Cellulase (Celluclast 1.5L) was kindly provided by Novozymes Biotechnology Co. Ltd (Guangzhou, China). Xylanase was purchased from Macklin Biochemical Co., Ltd (Shanghai, China). The enzyme activity of cellulase and xylanase was 753.52 EGU/g and 105 IU/g, respectively.
2.5.4. X-ray diffraction (XRD) The XRD patterns were measured using Rigaku-D/MAX instrument (Rigaku Corp., Tokyo, Japan) with Cu-Ka radiation generated at a voltage of 40 kV and a current of 30 mA with a 2θ range of 10–40° in steps of 0.02°. The crystallinity index (CrI) was calculated in accordance with the Segal method (without baseline substrate) (Segal et al., 1959).
2.2. Mixed enzymatic pretreatment 3. Results and discussion
Mixed enzyme pretreatment was carried out in 0.05 M sodium citrate buffer (pH 4.8) with a substrate loading of 1% (w/v) at 50 °C and 150 rpm for 2 h. The endoglucanase and xylanase loading were both 10–50 IU/g substrate during the pretreatment. After the reaction was completed, the mixed enzymes were denatured by incubating in water bath at 80 °C for 20 min. The pretreated pulp was then washed with deionized water to neutrality and placed at 4 °C. For simplicity, the resulting pretreated pulp were referred thereafter as H-10, H-30, H-50, L-10, L-30, and L-50.
3.1. Effects of mixed enzymatic pretreatment and post-treatment on the chemical compositions of LCNF Fig. 1 shows the chemical composition changes of LCNF produced from different processing stage. For comparison purpose, the results from original pulp (L or H) and corresponding LCNF produced using pure mechanical fibrillation were also presented. Because disk grinding was only a mechanical treatment, there were no obvious differences between the raw material and the resulting LCNF samples. Compared to the LCNF produced from mixed enzymatic pretreatment, the LCNF obtained by post-treatment contained lower percentage of carbohydrates (cellulose and hemicellulose) and higher percentage of lignin. This was because that LCNF was considerably fibrillated during the disk grinding and the surface area was increased, the mixed enzymes were easily adhered to fibril surface, resulting the significant degradation of carbohydrates. In addition, the mixed enzymatic post-treatment efficiency was less pronounced for H due to the presence of higher lignin content that had a negative effect on the efficiency of biological treatment and impeded carbohydrate degradation (Bian et al., 2018).
2.3. Mechanical fibrillation The original and mixed enzyme pretreated pulp were both distilled at a concentration of 1% (w/w), then mechanically fibrillated at 1500 rpm to produce LCNF using a stone disk grinder (Super Mass Collodier, Model: MKCA6-2 J, Disk Model: MKG-C, Masuko Sangyo Co., Ltd, Japan). The disk gap was first set to zero without pulp, and then with adjusted down to −200 µm. Fiber suspension was fed by gravity through a hopper and passed through the disk chamber for 20 times. 2.4. Mixed enzymatic post-treatment LCNF produced from original pulp was further post-treated using mixed enzyme. The posttreatment process was the same as the pretreatment described previously. The resultant LCNFs produced from mixed enzyme pretreatment and post-treatment were stored at 4 °C for further characterization.
3.2. Effects of mixed enzymatic pretreatment and post-treatment on the water retention value of LCNF Water retention value (WRV) is a measure of cellulose fibril water absorption and swelling ability, or the extent of fibrillation (Gu et al., 2018). H pulp with higher lignin content had lower WRV, as shown in Fig. 2, when compared to L pulp. This may be related to the existence of lignin that controlled water contents and shielded the free accessible hydroxyl group from the formation of hydrogen bonding with water molecules. It could be seen that the WRV was decreased obviously during mixed enzymatic post-treatment, which suggested that less water accessible cavities were established on fibers. From chemical composition point of view, mixed enzymes degraded large amounts of cellulose and hemicellulose of LCNF in the post-treatment, resulting in insufficient accessibility between fibril and water molecule. However, contrary to post-treatment, mixed enzymatic pretreatment removed partial carbohydrates and broke up the intermolecular and the intramolecular hydrogen bonds between hydroxyl groups, then subsequent mechanical fibrillation increased fibril internal surface and external surface area, allowing easier penetration of the water molecules between the fibrils to increase the WRV.
2.5. Analytic methods 2.5.1. Compositional analysis The chemical compositions (including structural polysaccharides and lignin) of the raw material and LCNF samples were determined using the National Renewable Energy Laboratory (NERL) standard method (Sluiter et al., 2008). 2.5.2. Water retention value The water retention value (WRV) stands for the total water in the pores of a substrate which closely be related to the total pore volume. The WRV of the original pulp fibers and all LCNF samples were measured and calculated according to the standard test method SCAN-C 62:00 in two replicates. As described in our previous work, the WRV is the percentage of retained water (weight change of the substrate before 2
Bioresource Technology 293 (2019) 122171
H. Bian, et al.
Fig. 2. Water retention value of unbleached pulp and corresponding LCNF samples. (a) Unbleached pulp with low lignin content (L) and corresponding LCNF samples; (b) Unbleached pulp with high lignin content (H) and corresponding LCNF samples. Fig. 1. Chemical compositions of two different unbleached pulp and corresponding LCNF samples. (a) Unbleached pulp with low lignin content (L) and corresponding LCNF samples; (b) Unbleached pulp with high lignin content (H) and corresponding LCNF samples.
number-average height of fibril became smaller as the mixed enzyme dosage increased, which suggested that higher enzyme dosage improved cellulose nanofibrillation efficiency.
3.3. Effects of mixed enzymatic pretreatment and post-treatment on the cellulose nanofibrillation efficiency
3.4. Effects of mixed enzymatic pretreatment and posttreatment on the crystallinity of LCNF
To compare the mixed enzymatic pretreatment and post-treatment on the cellulose nanofibrillation efficiency of LCNF. AFM imaging can provide meaningful measurements of fibril height that can be treated as a diameter (Supplementary data). The number-average height of LLCNF and H-LCNF were 97.8 and 109.8 nm, respectively. It was apparent that more uniform LCNF morphology could be obtained using both pretreatment and post-treatment, and the degree of nanofibrillation was more significant with number-average height of 15.0 nm (L50-LCNF) when mixed enzymes were added before mechanical fibrillation (Fig. 3). This was because that mixed enzymatic post-treatment mainly degraded carbohydrates and reduced cellulose chain length, however, pretreatment opened up the fibril structure with slightly sacrificing the carbohydrates composition, which was more beneficial for subsequent mechanical fibrillation. In addition, the
Besides WRV and fibril height, the crystallinity of LCNF is another crucial characteristic as the major indicator for cellulose nanofibrillation during LCNF production. The XRD patterns of both raw materials and all LCNF samples are presented (Supplementary data). Two main characteristic peaks at about 2θ = 16.4° and 22.6° corresponded to the (1 0 0) and (2 0 0) reflection planes, indicating that only cellulose I structure was present in all samples (Bian et al., 2019). These results suggest both mixed enzymatic pretreatment and post-treatment did not destroy or alter the inherent crystal structure of cellulose. Compared with the two original pulp fibers, crystallinity of all LCNF samples (except for H-30-LCNF and H-50-LCNF) produced from pure mechanical fibrillation with or without pretreatment or post-treatment were decreased (Fig. 4). These results suggested that mechanical fibrillation could break up substantial amounts of cellulose crystalline region, 3
Bioresource Technology 293 (2019) 122171
H. Bian, et al.
Fig. 3. Comparisons of the number-average fibril height of LCNF produced from two different unbleached pulps using enzymatic pretreatment and posttreatment.
Fig. 4. Crystallinity index of unbleached pulp and corresponding LCNF samples. (a) Unbleached pulp with low lignin content (L) and corresponding LCNF samples; (b) Unbleached pulp with high lignin content (H) and corresponding LCNF samples.
although mixed enzymes dissolved amorphous region, as revealed by the chemical compositions analysis.
Acknowledgements This work was supported by the National Key Research and Development Project of the 13th Five-Year Plan (2017YFD0601005) and the National Natural Science Foundation of China (Project No. 31470599). We would like to acknowledge Prof. Qiang Yong and Chenhuan Lai of Nanjing Forestry University for complimentarily providing us the xylanase. We also would like to acknowledge Dr. Chen Huang of China Institute of Chemical Industry of Forestry Products for giving valuable guidance on the use of XRD.
4. Conclusion In this study, mixed enzymatic pretreatment and post-treatment were employed to produce lignocellulosic nanofibrils, and the cellulose nanofibrillation efficiency along with the LCNF properties were compared. It appeared that mixed enzymatic pretreatment was more suitable than post-treatment for enhancing the cellulose nanofibrillation, based on the resulting LCNF with higher water retention value, higher crystallinity and smaller height. Meanwhile, the entire process was environmentally friendly and did not change the crystal structure of cellulose. Overall, this work demonstrates that enzymatic pretreatment before mechanical fibrillation is a more efficient biological treatment to facilitate nanocellulose production and application.
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biortech.2019.122171.
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References
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Beltramino, F., Roncero, M.B., Torres, A.L., Vidal, T., Valls, C., 2016. Optimization of sulfuric acid hydrolysis conditions for preparation of nanocrystalline cellulose from enzymatically pretreated fibers. Cellulose 23 (3), 1777–1789. Bian, H., Chen, L., Dai, H., Zhu, J.Y., 2017. Integrated production of lignin containing cellulose nanocrystals (LCNC) and nanofibrils (LCNF) using an easily recyclable dicarboxylic acid. Carbohydr. Polym. 167, 167–176. Bian, H., Gao, Y., Luo, J., Jiao, L., Wu, W., Fang, G., Dai, H., 2019. Lignocellulosic nanofibrils produced using wheat straw and their pulping solid residue: from agricultural waste to cellulose nanomaterials. Waste Manage. 91, 1–8. Bian, H., Gao, Y., Yang, Y., Fang, G., Dai, H., 2018. Improving cellulose nanofibrillation of waste wheat straw using the combined methods of prewashing, p-toluenesulfonic acid hydrolysis, disk grinding, and endoglucanase post-treatment. Bioresour. Technol. 256, 321–327. Gu, F., Wang, W.X., Cai, Z.S., Xue, F., Jin, Y.C., Zhu, J.Y., 2018. Water retention value for characterizing fibrillation degree of cellulosic fibers at micro and nanometer scales. Cellulose 25 (5), 2861–2871. Hu, J., Tian, D., Renneckar, S., Saddler, J.N., 2018. Enzyme mediated nanofibrillation of cellulose by the synergistic actions of an endoglucanase, lytic polysaccharide monooxygenase (LPMO) and xylanase. Sci. Rep. 8 (1), 3195. Jiang, F., Hsieh, Y.-L., 2016. Self-assembling of TEMPO oxidized cellulose nanofibrils as affected by protonation of surface carboxyls and drying methods. ACS Sustain. Chem. Eng. 4 (3), 1041–1049. Lavoine, N., Desloges, I., Dufresne, A., Bras, J., 2012. Microfibrillated cellulose – its barrier properties and applications in cellulosic materials: a review. Carbohydr. Polym. 90 (2), 735–764. Long, L., Tian, D., Hu, J., Wang, F., Saddler, J., 2017. A xylanase-aided enzymatic pretreatment facilitates cellulose nanofibrillation. Bioresour. Technol. 243, 898–904. Long, L., Tian, D., Zhai, R., Li, X., Zhang, Y., Hu, J., Wang, F., Saddler, J., 2018. Thermostable xylanase-aided two-stage hydrolysis approach enhances sugar release
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Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Comparison of mixed enzymatic pretreatment and post-treatment for enhancing the cellulose nanofibrillation efficiency
T
Huiyang Biana,b, Maolin Dongb, Lidong Chenb, Xuelian Zhoua,b, Shuzhen Nia,b,c, Guigan Fangd, ⁎ Hongqi Daia,b, a
Jiangsu Co-Innovation Center of Efficient Processing and Utilization of Forest Resources, Nanjing Forestry University, Nanjing 210037, China College of Light Industry and Food Engineering, Nanjing Forestry University, Nanjing 210037, China c State Key Laboratory of Biobased Material and Green Papermaking, Qilu University of Technology, Shandong Academy of Sciences, Jinan 250353, China d China Institute of Chemical Industry of Forestry Products, Chinese Academy of Forestry, Nanjing 210042, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Endoglucanase Xylanase Enzymatic pretreatment Enzymatic post-treatment Cellulose nanofibrillation
In this work, lignocellulosic nanofibrils (LCNF) produced from mechanical fibrillation with mixed enzymatic pretreatment or post-treatment were compared and the chemical composition, water retention value (WRV), average-number height and crystallinity for the obtained LCNF were evaluated. Compared to pure mechanical fibrillation, both mixed enzymatic pretreatment and post-treatment could efficiently facilitate cellulose nanofibrillation. Moreover, mixed enzymatic pretreatment was more suitable for LCNF production, resulting in a relatively higher WRV of 909% and smaller average-number height of 15 nm. These discoveries provide new insights into a more efficient biological method for the production and application of cellulose nanomaterials.
1. Introduction Lignocellulosic biomass, as the most abundant carbon-neutral resource on the earth, is considered as a sustainable raw material used for the production of different high-value added products (e.g., cellulose nanomaterials, biofuels and chemicals) (Zhu et al., 2011). One promising new product is cellulose nanofibrils (CNF), which can be used in applications such as hydrogels, supercapacitors, biosensors and energy storage devices due to its high elastic modulus, high specific surface area, and low thermal expansion coefficient (Siró and Plackett, 2010). The most commonly used method to produce CNF is via pure mechanical fibrillation equipment including grinders, homogenizers and microfluidizers (Lavoine et al., 2012). However, the high energy consumption during mechanical treatment and the heterogeneity of products still limited its implementation (Nair et al., 2014). In order to address these issues, chemical or biological treatments has been employed to “open up/loose” the intrinsic recalcitrance of cellulosic fibers, thus decreasing the energy required for the fibrillation (Wang et al., 2018). Although various chemical pretreatment approaches (e.g., TEMPO-oxidation, carboxylation and sulfonation) could achieve the goal, the severe corrosion of equipment and the difficulties to recycle chemicals have limited their further application (Beltramino et al., 2016; Jiang and Hsieh, 2016; Wang et al., 2017). Comparing to the
⁎
chemical pretreatment approaches, biological treatment is considered to be more attractive due to the low enzyme loading, high selectivity of enzymatic treatment and the environmentally friendly reaction conditions. To date, several types of enzymatic pretreatments have been investigated to improve the cellulose nanofibrillation. Most of the works were focused on using canonical endoglucanase (EG), which could randomly cleavage the cellulose β-1,4 linkages at less organized region (Pääkkö et al., 2007). Other so-called auxiliary enzymes, such as hemicellulases, laccases and lytic polysaccharide monooxygenases (LPMO), even though did not directly hydrolyze cellulose, could selectively remove hemicellulose and lignin from the carbohydrate networks (Hu et al., 2018; Long et al., 2018). In addition, the synergistic cooperation between cellulases and xylanases could greatly open up the cellulosic fiber without sacrificing cellulose crystal structure and significantly enhance the cellulose nanofibrillation (Long et al., 2017; Tian et al., 2017). Recently, we and others have found endoglucanase posttreatment also could produce less entangled fibrils with thinner diameters (Bian et al., 2018; Wang et al., 2016). However, there are no publicly available researches focused on comparing the cellulose nanofibrillation efficiency using mixed enzymatic pretreatment and posttreatment. In this work, unbleached mixed hardwood pulp was pretreated or
Corresponding author. E-mail address: [email protected] (H. Dai).
https://doi.org/10.1016/j.biortech.2019.122171 Received 10 September 2019; Received in revised form 17 September 2019; Accepted 18 September 2019 Available online 20 September 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
Bioresource Technology 293 (2019) 122171
H. Bian, et al.
and after drying) to the dried substrate (Bian et al., 2017).
post-treated by two types of enzymes (endoglucanase and xylanase) to produce lignocellulosic nanofibrils (LCNF). Several physicochemical characteristics such as chemical composition, water retention value, fibril morphology and crystallinity were investigated to compare the mixed enzyme pretreatment and post-treatment on the cellulose nanofibrillation efficiency and LCNF properties.
2.5.3. Morphology analysis The morphologies of LCNF samples produced from mixed enzyme pretreatment and post-treatment were analyzed by atomic force microscopy (AFM, Dimension Edge, Bruker, Germany). Samples were diluted to solids consistency of 0.01 wt% and deposited onto clean mica substrates and air dried overnight at room temperature. AFM topographical images were obtained in tapping mode at 300 kHz using a standard silicon cantilever and a tip with radius of curvature of 8 nm. Fibril height distribution was measured using Gwyddion software (Department of Nanometrology, Czech Metrology Institute, Crezh Republic, 64-bit) and number-average fibril heights were calculated.
2. Materials and methods 2.1. Materials Two unbleached mixed hardwood pulps with lignin content of 17.2% and 3.9%, defined as H and L, were complimentarily provided from International Paper Company (Loveland, OH). These two pulp samples were acquired from the same mill but different production lines. Cellulase (Celluclast 1.5L) was kindly provided by Novozymes Biotechnology Co. Ltd (Guangzhou, China). Xylanase was purchased from Macklin Biochemical Co., Ltd (Shanghai, China). The enzyme activity of cellulase and xylanase was 753.52 EGU/g and 105 IU/g, respectively.
2.5.4. X-ray diffraction (XRD) The XRD patterns were measured using Rigaku-D/MAX instrument (Rigaku Corp., Tokyo, Japan) with Cu-Ka radiation generated at a voltage of 40 kV and a current of 30 mA with a 2θ range of 10–40° in steps of 0.02°. The crystallinity index (CrI) was calculated in accordance with the Segal method (without baseline substrate) (Segal et al., 1959).
2.2. Mixed enzymatic pretreatment 3. Results and discussion
Mixed enzyme pretreatment was carried out in 0.05 M sodium citrate buffer (pH 4.8) with a substrate loading of 1% (w/v) at 50 °C and 150 rpm for 2 h. The endoglucanase and xylanase loading were both 10–50 IU/g substrate during the pretreatment. After the reaction was completed, the mixed enzymes were denatured by incubating in water bath at 80 °C for 20 min. The pretreated pulp was then washed with deionized water to neutrality and placed at 4 °C. For simplicity, the resulting pretreated pulp were referred thereafter as H-10, H-30, H-50, L-10, L-30, and L-50.
3.1. Effects of mixed enzymatic pretreatment and post-treatment on the chemical compositions of LCNF Fig. 1 shows the chemical composition changes of LCNF produced from different processing stage. For comparison purpose, the results from original pulp (L or H) and corresponding LCNF produced using pure mechanical fibrillation were also presented. Because disk grinding was only a mechanical treatment, there were no obvious differences between the raw material and the resulting LCNF samples. Compared to the LCNF produced from mixed enzymatic pretreatment, the LCNF obtained by post-treatment contained lower percentage of carbohydrates (cellulose and hemicellulose) and higher percentage of lignin. This was because that LCNF was considerably fibrillated during the disk grinding and the surface area was increased, the mixed enzymes were easily adhered to fibril surface, resulting the significant degradation of carbohydrates. In addition, the mixed enzymatic post-treatment efficiency was less pronounced for H due to the presence of higher lignin content that had a negative effect on the efficiency of biological treatment and impeded carbohydrate degradation (Bian et al., 2018).
2.3. Mechanical fibrillation The original and mixed enzyme pretreated pulp were both distilled at a concentration of 1% (w/w), then mechanically fibrillated at 1500 rpm to produce LCNF using a stone disk grinder (Super Mass Collodier, Model: MKCA6-2 J, Disk Model: MKG-C, Masuko Sangyo Co., Ltd, Japan). The disk gap was first set to zero without pulp, and then with adjusted down to −200 µm. Fiber suspension was fed by gravity through a hopper and passed through the disk chamber for 20 times. 2.4. Mixed enzymatic post-treatment LCNF produced from original pulp was further post-treated using mixed enzyme. The posttreatment process was the same as the pretreatment described previously. The resultant LCNFs produced from mixed enzyme pretreatment and post-treatment were stored at 4 °C for further characterization.
3.2. Effects of mixed enzymatic pretreatment and post-treatment on the water retention value of LCNF Water retention value (WRV) is a measure of cellulose fibril water absorption and swelling ability, or the extent of fibrillation (Gu et al., 2018). H pulp with higher lignin content had lower WRV, as shown in Fig. 2, when compared to L pulp. This may be related to the existence of lignin that controlled water contents and shielded the free accessible hydroxyl group from the formation of hydrogen bonding with water molecules. It could be seen that the WRV was decreased obviously during mixed enzymatic post-treatment, which suggested that less water accessible cavities were established on fibers. From chemical composition point of view, mixed enzymes degraded large amounts of cellulose and hemicellulose of LCNF in the post-treatment, resulting in insufficient accessibility between fibril and water molecule. However, contrary to post-treatment, mixed enzymatic pretreatment removed partial carbohydrates and broke up the intermolecular and the intramolecular hydrogen bonds between hydroxyl groups, then subsequent mechanical fibrillation increased fibril internal surface and external surface area, allowing easier penetration of the water molecules between the fibrils to increase the WRV.
2.5. Analytic methods 2.5.1. Compositional analysis The chemical compositions (including structural polysaccharides and lignin) of the raw material and LCNF samples were determined using the National Renewable Energy Laboratory (NERL) standard method (Sluiter et al., 2008). 2.5.2. Water retention value The water retention value (WRV) stands for the total water in the pores of a substrate which closely be related to the total pore volume. The WRV of the original pulp fibers and all LCNF samples were measured and calculated according to the standard test method SCAN-C 62:00 in two replicates. As described in our previous work, the WRV is the percentage of retained water (weight change of the substrate before 2
Bioresource Technology 293 (2019) 122171
H. Bian, et al.
Fig. 2. Water retention value of unbleached pulp and corresponding LCNF samples. (a) Unbleached pulp with low lignin content (L) and corresponding LCNF samples; (b) Unbleached pulp with high lignin content (H) and corresponding LCNF samples. Fig. 1. Chemical compositions of two different unbleached pulp and corresponding LCNF samples. (a) Unbleached pulp with low lignin content (L) and corresponding LCNF samples; (b) Unbleached pulp with high lignin content (H) and corresponding LCNF samples.
number-average height of fibril became smaller as the mixed enzyme dosage increased, which suggested that higher enzyme dosage improved cellulose nanofibrillation efficiency.
3.3. Effects of mixed enzymatic pretreatment and post-treatment on the cellulose nanofibrillation efficiency
3.4. Effects of mixed enzymatic pretreatment and posttreatment on the crystallinity of LCNF
To compare the mixed enzymatic pretreatment and post-treatment on the cellulose nanofibrillation efficiency of LCNF. AFM imaging can provide meaningful measurements of fibril height that can be treated as a diameter (Supplementary data). The number-average height of LLCNF and H-LCNF were 97.8 and 109.8 nm, respectively. It was apparent that more uniform LCNF morphology could be obtained using both pretreatment and post-treatment, and the degree of nanofibrillation was more significant with number-average height of 15.0 nm (L50-LCNF) when mixed enzymes were added before mechanical fibrillation (Fig. 3). This was because that mixed enzymatic post-treatment mainly degraded carbohydrates and reduced cellulose chain length, however, pretreatment opened up the fibril structure with slightly sacrificing the carbohydrates composition, which was more beneficial for subsequent mechanical fibrillation. In addition, the
Besides WRV and fibril height, the crystallinity of LCNF is another crucial characteristic as the major indicator for cellulose nanofibrillation during LCNF production. The XRD patterns of both raw materials and all LCNF samples are presented (Supplementary data). Two main characteristic peaks at about 2θ = 16.4° and 22.6° corresponded to the (1 0 0) and (2 0 0) reflection planes, indicating that only cellulose I structure was present in all samples (Bian et al., 2019). These results suggest both mixed enzymatic pretreatment and post-treatment did not destroy or alter the inherent crystal structure of cellulose. Compared with the two original pulp fibers, crystallinity of all LCNF samples (except for H-30-LCNF and H-50-LCNF) produced from pure mechanical fibrillation with or without pretreatment or post-treatment were decreased (Fig. 4). These results suggested that mechanical fibrillation could break up substantial amounts of cellulose crystalline region, 3
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Fig. 3. Comparisons of the number-average fibril height of LCNF produced from two different unbleached pulps using enzymatic pretreatment and posttreatment.
Fig. 4. Crystallinity index of unbleached pulp and corresponding LCNF samples. (a) Unbleached pulp with low lignin content (L) and corresponding LCNF samples; (b) Unbleached pulp with high lignin content (H) and corresponding LCNF samples.
although mixed enzymes dissolved amorphous region, as revealed by the chemical compositions analysis.
Acknowledgements This work was supported by the National Key Research and Development Project of the 13th Five-Year Plan (2017YFD0601005) and the National Natural Science Foundation of China (Project No. 31470599). We would like to acknowledge Prof. Qiang Yong and Chenhuan Lai of Nanjing Forestry University for complimentarily providing us the xylanase. We also would like to acknowledge Dr. Chen Huang of China Institute of Chemical Industry of Forestry Products for giving valuable guidance on the use of XRD.
4. Conclusion In this study, mixed enzymatic pretreatment and post-treatment were employed to produce lignocellulosic nanofibrils, and the cellulose nanofibrillation efficiency along with the LCNF properties were compared. It appeared that mixed enzymatic pretreatment was more suitable than post-treatment for enhancing the cellulose nanofibrillation, based on the resulting LCNF with higher water retention value, higher crystallinity and smaller height. Meanwhile, the entire process was environmentally friendly and did not change the crystal structure of cellulose. Overall, this work demonstrates that enzymatic pretreatment before mechanical fibrillation is a more efficient biological treatment to facilitate nanocellulose production and application.
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biortech.2019.122171.
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